Thyroid diseases are commonly caused by abnormal thyroid cell proliferation and differentiation, which can lead to various, possibly fatal complications. The mechanisms by which these diseases develop by are largely unknown and have yet to be discovered. Presently, the long-term goal of our parent grant is to elucidate the molecular mechanisms by which embryonic stem (ES) cells differentiate into definitive thyrocytes. Pluripotent ES cells, derived from the inner cell mass of early embryos, can both self-renew and differentiate into all cell types in the body and as such provide a key thyroid developmental model. The manipulation of the pluripotent potential of murine ES cells in vitro has allowed us to direct differentiation towards the thyroid lineage under the appropriate conditions. To expand upon this mouse model, we are also working to develop a similar model using NIH-registry human ES cells. We have recently developed a form of induced pluripotent stem (iPS) cell technology, which can reprogram adult skin fibroblasts into an ES cell-like state using the retrovirus-mediated transfection of four transcription factors (Oct4, Sox2, c-Myc, and Klf4). Our manipulated iPS cells are similar to ES cells in many respects, including the expression of main stem cell genes and proteins, embryoid body formation, teratomas formation, potency and differentiation. In this application, we will expand on human ES cell studies focused on the mechanisms regulating endoderm induction and its specification to thyroid lineage during ES cell differentiation. Furthermore, we propose to establish and propagate murine iPS cells from a mutant TSHR hypothyroid mouse model. The hypothyroid mice display severe congenital hypothyroidism and thyroid hyperplasia and provide an opportunity to explore the pathophysiological manifestations of this disease. The goal of Specific Aim 1 is to generate thyroid follicular cells from human ES cells. The goal of Specific Aim 2 is to produce and characterize murine iPS cells from normal and mutant TSHR fibroblasts. We will use our established methods to reprogram skin fibroblasts of normal and mutant TSHR mice into iPS cells by the ectopic expression of four transcription factors. We will test the hypothesis that the disease-specific iPS cells can be coaxed into thyroid follicular cells to explore the biological processes that lead to disease phenotypes. Finally, we will characterize the thyrocyte differentiation potential of these murine iPS cell lines in comparison to our well-established murine TSHR-/- ES cell lines by using in vitro and in vivo assays. More specifically, to initiate effort to new therapeutic approaches, we will use these disease-iPS cells as assay tools to validate the defects of iodide transport and thyroid hormone synthesis and secretion. The success of these aims will provide a number of therapeutic promises including: 1) the ability to establish patient-derived iPS cell lines as a research tool to model human disease, and 2) the opportunity to use patient-derived iPS cell lines to perform sophisticated testing of candidate therapeutics and screening assays.